Tissue specific patterns of 5-methylcytosine residues in vertebrate DNAs are thought to play a role in controlling gene expression and these patterns can be inherited following DNA replication. Some of the details of the mechanisms governing the inheritance of modification patterns are understood, but virually nothing is known of how methyl groups are lost from DNA during cellular differentiation. This grant will determine how the application of methyl groups to DNA is controlled through S phase and investigate possible mechanisms for demethylation. The cell cycle kinetics of DNA methylation relative to DNA synthesis and the effects of macromolecular synthesis inhibitors on these processes will be determined. The methylation of DNA made under repair conditions in G1 cells will be contrasted with the synthesized in S phase. The cell cycle control of DNA methyltransferase expression and aggregation will be investigated. The abilities of nuclear constituents (histones and non-histone proteins and RNA) to prevent the in vitro methylation of hemimethylated duplex DNA will be studied. Such inhibition may explain how failure to methylate in critical cell cycles may lead to symmetrical demethylation of specific DNA sequences. Finally, a search will be made for demethylase enzymes capable of the removal of methyl groups from symmetrically or hemimethylated sites.